The aim of the present study was to investigate morphohistochemical and electron-microscopic peculiar features of DC generated from bone marrow precursor cells of mice.
Bone marrow of CBA line mice (20 experimental animals) was homogenized and transferred into full culture medium containing granulocytic-macrophagal colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). At the 6th day, medium was substituted with addition of tumor necrosis factor (TNF-α - 100 ng/ml) to induce DC maturation and on the 9th day dendritic cells obtained were collected. Smears obtained from cell suspension were subjected to Romanovskiy-Gimza´s staining with azur II and eosin, Brashe´s staining with methyl green and pyronine, Shabadash´s control by RNA and Shiff-reagent using amylase. Electron-microscopic preparations were examined and photographed with electron microscope JEM-100CX (LEOL, Japan).
As a result of coincubation of bone marrow precursor cells with GM-CSF and IL-4, immature dendritic cells were obtained during 6 days, mature dendritic cells after TNF- α pulsation - at the 9th day. It was confirmed by the data of immunophenotype of generated cells.
Morphohistochemical studies have shown that dendritic cells are large cells with eccentrically located nucleus containing nucleoli, vacuolized cytoplasm and typical processes on the surface. Romanovskiy-Gimza´s staining of cytoplasm was weakly basophilic in immature DC and intensively basophilic in mature ones. Brashe´s staining gave more pronounced cytoplasm pyroninophilia in mature DC forms than in immature ones that demonstrated higher RNA content. Shiff-reagent treatment revealed Shiff iodine acid positive granules in cytoplasm (more numerous and larger in mature cells) whose intensity of staining was significantly reduced after amylase effect in immature DC and practically did not change in mature ones. It proves presence of both glycosaminoglycanes and glycogen with the latter predominating in immature types of dendritic cells.
Electron-microscopic investigations have shown that dendritic cells generated from bone marrow precursor cells were large in their size, had oval or irregular form with numerous branched and pin-shaped processes, eccentrically located nucleus with numerous invaginations, chromatine concentrated mainly in periphery and large nucleoli. Cytoplasm contained a great number of vesicles of various size and vacuoles with varied content. In DC cytoplasm synthetic organoids such as mitochondria, smooth and granular endoplasmic network, ribosomes and Golgi apparatus were well developed.
Thus, our investigations have demonstrated the possibility of obtaining DC from mouse bone marrow precursor cells with cytokine stimulation. The results obtained have confirmed the data on the possibility of directed differentiation of bone marrow precursor cells into specialized cellular types under the effect of growth factors. Dendritic cells generated with cytokines can serve as a basis for production of DC-vaccines which can be applied in biotherapy of oncologic and infectious diseases.
Библиографическая ссылкаLebedinskaya O.V., Melekhin S.V., Lebedinskaya E.A., Kiselevskiy M.V. MORPHOHISTOCHEMICAL AND ELECTRON-MICROSCOPIC CHARACTERISTIC OF DENDRITIC CELLS GENERATED FROM BONE MARROW PRECURSOR CELLS // Успехи современного естествознания. – 2006. – № 8. – С. 45-45;
URL: http://www.natural-sciences.ru/ru/article/view?id=11221 (дата обращения: 21.10.2019).